The serine/threonine protein kinase Akt, also known as protein kinase B (PKB) or RAC-PK, was initially identified as one of the downstream targets of phosphatidylinositol-3 kinase (PI3K). Activated Akt plays a key role in mediating signals for cell growth, cell survival (anti-apoptotic), cell-cycle progression, differentiation, transcription, translation and glucose metabolism.
The Akt pathway is damaged in skeletal muscles of amyotrophic lateral sclerosis (ALS) patients, as in SOD1 mice (a mice model for ALS). Although no difference in Akt mRNA levels is found in ALS patients when compared to control subjects, at the protein level, ALS patients, have a significantly lower content of the active phosphorylated Akt protein in comparison to healthy control subjects (Leger et al., FASEB J. 2006; 20(3):583-585). Akt pathway dysfunction has also been shown in motoneurons of both sporadic and familial ALS patients (Dewil et al., Neuropathol Appl Neurobiol., 2007; 33(5):499-509).
The peptides LPPLPYP (SEQ ID NO: 1; also known as Stressin-1) and PYPLPPL (SEQ ID NO: 2, where all residues are in the “D” isomeric form) were first disclosed in WO 2006/021954, where their efficacy in ameliorating stress-induced cell death and p53-mediated response was demonstrated. According to the disclosure, these peptides are also useful in treating inflammatory and autoimmune diseases. Nowhere in the background art was it taught or suggested that the activity of peptides comprising SEQ ID NO: 1 or SEQ ID NO: 2 or derivatives thereof is affected by the level of Akt and Akt phosphorylation, such that, the therapeutic effect of these peptides would be significantly higher in subjects having low levels of pAkt and, optionally, low levels of pAkt:tAkt ratio. In addition, the background art does not teach or even suggests the use of pAkt and pAkt:tAkt ratio as effective markers for ALS and moreover for staging the progression of ALS in ALS patients.
ALS is a devastating and rapidly fatal disease with currently only one available, FDA-approved, modestly effective treatment. The approve therapy, Rilutek® (Riluzole), has a modest benefit estimated to be a three month extension in patient survival.
There is therefore an urgent need for new therapies. Recent attempts to find molecules that could provide a beneficial therapy for ALS include the finding of 1,4-Diaza-bicyclo[3.2.2]non-6-en-4-yl)-heterocyclyl-methanone ligands disclosed in US Patent Application, Publication No. 2010/0298306. The ligands are directed to treatment of any Nicotinic Acetylcholine Receptors, inter alia, ALS. In addition, US Patent Application, Publication No. 2010/0099700, discloses use of hydrogenated pyrido(4,3-b)indole for treating ALS. US Patent Application, Publication No. 2009/0324549 discloses methods for treating ALS comprising administration to a patient in need thereof, proteins and/or peptides characterized in that they originate from the gene which results from the retention of the intron 3 of the gene SMN (or survival motor neuron) identified in the gene bank with the access number AY876898.
WO 2011/017030 discloses a method of treating a disease associated with excess activation of monocytes to activated macrophages, including, inter alia, ALS said method comprising administering a therapeutically-effective amount of an oxidative agent to a subject in need thereof, wherein said oxidative agent is selected from the group consisting of non-halogen activated-oxygen compounds, non-oxygen activated-halogen compounds, and N-halo compounds. According to WO 2011/017030 the non-halogen activated-oxygen compounds are selected from potassium nitrate (KNO3), permanganate salts, ammonium cerium(IV) nitrate, hexavalent chromium compounds, chromate/dichromate compounds, ammonium silver nitrate, sulfoxides, persulfuric acid, osmium tetroxide (OsO4), nitric acid, nitrous oxide (N2O), hydrogen peroxide, organic peroxides, superoxides, and ozone; the non-oxygen activated-halogen compounds are selected from fluorine, chlorine, bromine, and iodine; the N-halo compounds are selected from the group consisting of N-halophthalimide, N-halosuccinimide, N-halosaccharin, N,N-dihalourethane, N-haloacetanilide, 1,3-dihalo-5,5-dimethylhydantoin, trihaloisocyanuric acid and sodium dihaloisocyanurate; and the oxidative agent is selected from 1,3-dichloro-5,5-dimethylhydantoin and chloramine-T.
The development of the first genetically based mouse model of ALS in 1994, energized the field of preclinical testing despite numerous unforeseen complexities along the way. Transgenic mutant SOD1 mice, the only ALS mouse models currently available, have mutations in the Cu/Zn Superoxide Dismutase 1 gene (SOD1) which account for ˜20% of Familial ALS (FALS) cases, corresponding to 2-3% of all ALS cases. Transgenic mutant SOD1 mice exhibit all of the histopathological hallmarks observed clinically in sporadic and familial ALS.
Because there is no obvious mutational hotspot and no clear correlation between the level of enzymatic activity of the mutant SOD1 protein and the observed disease phenotype or clinical progression, SOD1 is thought to act primarily via a toxic gain of function in ALS, although loss of function may also contribute to disease pathophysiology. It is generally thought that the different mutant SOD1 proteins are likely to cause ALS by a similar mechanism.
Several transgenic mouse models have been generated to model mutations found in FALS patients. In all of these mouse models, massive death of motor neurons in the ventral horn of the spinal cord and loss of myelinated axons in ventral motor roots ultimately leads to paralysis and muscle atrophy. All of these mouse models have been reported to exhibit the same histopathological hallmarks associated with ALS in humans: progressive accumulation of detergent-resistant aggregates containing SOD1 and ubiquitin and aberrant neurofilament accumulations in degenerating motor neurons. In addition to neuronal degeneration, reactive astroglia and microglia have also been detected in diseased tissue in the mice, similar to that observed in humans.
Despite these histopathological similarities, the timing of onset and rate of disease progression differ (often dramatically) among the various SOD1 transgenic mouse models.
ALS is commonly assessed by neurological score and weight loss. As used herein, the term “neurological score” and “neurologic score” are interchangeably used herein to describe the common standards for assessing the presence or stage of a neurologic disease, such as ALS. Some example of commonly used neurological scoring systems include: measurements of splay (or other measures of paralysis) and beam walk.
In SOD1 mice, regardless of which neurological scoring system is used, scores are typically assessed for both hind legs of the SOD1 mice. The example neurological scoring system below employs a scale of zero to four researchals(dot)org/uploaded_files/p41_jax_sod1manual_20091202_29aPcx(dot)pdf. Example of score criteria used to assign each score under this system are as follows:                Score of 0: full extension of hind legs away from lateral midline when mouse is suspended by its tail, and mouse can hold this for two seconds, suspended two to three times.        Score of 1: collapse or partial collapse of leg extension towards lateral midline (weakness) or trembling of hind legs during tail suspension.        Score of 2: toes curl under at least twice during walking of 12 inches, or any part of foot is dragging along cage bottom/table.        Score of 3: rigid paralysis or minimal joint movement, foot not being used for generating forward motion.        Score of 4: mouse cannot right itself within 30 seconds after being placed on either side.        
U.S. Pat. No. 7,659,243 discloses the use of angiogenin, or a fragment or variant thereof, to treat diseases characterized by neuronal injury or death, or axonal degeneration, especially neurodegenerative diseases such as ALS. According to the disclosure, the neuroprotective effect of angiogenin involves the activation of the PI3K/Akt pathway.
U.S. Pat. Nos. 7,030,090 and 7,517,857 disclose a peptide that stimulates Akt phosphorylation via activation of formyl peptide receptor or formyl peptide receptor-like 1, the peptide comprises an amino acid sequence of WX1X2MX3X4, where X1=K, R, E, H or D, X2=G, Y, H, E or W, X3=V or G and X4=D-Me or G.
U.S. Pat. No. 7,622,455 discloses a method of treating ALS comprising administering to the cerebrospinal fluid of a subject in need thereof antisense oligonucleotides complementary to SOD1 nucleic acids.
There is an unmet need for novel methods for slowing down the rate of progression of neurodegenerative diseases, such as ALS, assessing responsiveness to treatment of the disease, and staging the disease in a manner that is specific, safe and effective.